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Microbial Genetics

Microbial Genetics. WHY? terms Genotype refers to genetic makeup Phenotype refers to expression of that genetic makeup Heritable traits must be encoded in DNA Mutations occur randomly. Mutants resistant to an antibiotic. Kinds of mutants. Auxotroph : loss of biosynthetic enzyme

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Microbial Genetics

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  1. Microbial Genetics • WHY? • terms • Genotype refers to genetic makeup • Phenotype refers to expression of that genetic makeup • Heritable traits must be encoded in DNA • Mutations occur randomly

  2. Mutants resistant to an antibiotic

  3. Kinds of mutants • Auxotroph: loss of biosynthetic enzyme • Cold- or temperature-sensitive: enzyme function is restricted to narrower temperature range • Drug resistance or sensitivity: permeability, degradation, or site of action • Morphology: change in capsule or other feature leads to change in colony • Catabolic: loss of a degradative enzyme leads to, for example, inability to ferment a carbohydrate • Virus-resistant: loss of surface feature that is a virus receptor

  4. Results of point mutations

  5. Frame-shift mutations

  6. Reversions • Same-site reversion: may be true revertant (same sequence) or just restore activity • Second-site reversion • Frame-shift • Production of another enzyme that fulfills the function

  7. Mutagens • Analogs for bases • 5-bromo-uracil for thymine (5BU can pair with G as well as with A) • 2-aminopurine for adenine (2AP can pair with C as well as with T) • Intercalating agents (ethidium bromide) • UV (260 nm) causes dimerization of adjacent thymines (photoreactivation) • Ionizing radiation causes chemical changes to the DNA • Generation of free radicals that can react with DNA • SS or DS breaks in the DNA molecule • Transposable genetic elements

  8. Recombination

  9. Recombination

  10. Genetic exchange: transformation

  11. Genetic exchange: plasmid transfer • Small, usually circular, independently replicating DNA molecules • Generally, G- plasmids replicate as does chromosomal DNA, G+ plasmids by “rolling circle” replication • Genes of replication control, timing initiation on plasmid (ori) • Some plasmids integrate (F+, Hfr) • Most are double-stranded • About 1- 100kb • Code for: • R-factors (R-plasmids) : antibiotic resistance, heavy metal resistance • Virulence plasmids : adhesins, hemolytic factors, toxin, Ti, bacteriocins • Degradation, tol, nah, • Plasmid copy # • Compatibility (inc)

  12. Avery Experiment

  13. Transformation • Competence: ability to be transformed • Steps in transformation • DNA binding and uptake (SS or DS, depending on species) • Integration (recA) • Competence may be induced by electroporation, Ca

  14. Mechanism of transformation

  15. Transduction • Generalized—can be carried out by either lytic or temperate phage • Specialized—requires specific integration

  16. Generalized transduction

  17. Specialized transduction

  18. Specialized transduction

  19. Phage Conversion • Lysogeny may cause other changes in the host cell • Often the host acquires immunity to additional infection by that phage type • There may be other changes that may be beneficial to the host • Lysogenized Salmonella anatum acquires cell-surface changes • Lysogenized Corynebacterium diphtherium acquires toxin

  20. Genetic exchange: conjugation

  21. Conjugation: early Cell-surface structure

  22. Conjugation: middle

  23. Conjugation: late

  24. Transposons • Rare events • Mobile Genetic elements-”jumping genes” • Carry a transposase, and flanked by inverted repeats • 20bp to >100bp • First discovered in maize/corn • Phase variation (invertible elements, need invertase)

  25. Transposons

  26. Genetic Engineering-basics • Basic steps in cloning • Restriction-modification enzymes • “shot gun” or PCR • Ligase • Recombination Plasmids (or phage)= vectors • Expression vectors • Selection of clones • Looking for a clone with a specific gene • Probes (DNA, RNA or antibody) • Wave of the future: DNA chips or “microarrays”, BAC libraries, automated sequencing etc

  27. Genomics • Bioinformatics • Harvesting genes for biotech (Diversa) • Recent Science article • Comparing gene families

  28. Escherichia coli map

  29. Next Tues • NO quiz • Chp 12, finish up where we left of Today

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